gender: male age (years): 38 status: smoker spermatozoa (m/ml): 89 round cells (m/ml): 1.8 motility (a/b/c/d): 20/30/20/30 % normal forms: 26 cigarette/day (starting year): 10 (1987)
Extracted molecule
total RNA
Extraction protocol
Subsequent to storage, samples were thawed and washed twice in 2 ml of PBS, then suspended in round cell lysis buffer (0.1% SDS, 0.5% Triton X-100 in RNase free H2O). RNA was extracted using the RNeasy mini kit (Qiagen GmbH., Germany) with gentle modifications. Sperm cells were suspended in 600 µl of lysis buffer for 107 cells. This cell suspention was lysed using a Politron as previously described (Saade, et al., 2007), to maximize the recovery of spermatozoal RNA. The lysates were then processed according to the manufacturer’s instructions using the animal cell protocol. After RNase-free DNase treatment (Qiagen GmbH., Germany), the RNA was eluted with 30µl. RNA concentration was determined using a nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA integrity was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared according to the miRNA Microarray System with miRNA Complete labelling.
Hybridization protocol
Cy3-labelled cRNA was hybridize using the manufacter's protocole (miRNA Microarray System with miRNA Complete labelling and Hyb kit).
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
Description
S3 Tobacco-induced gene expression alterations in human spermatozoa
Data processing
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol miRNA_107_Sept09_patch and Grid: US83700202_253118111117_S01_grid) to obtain background subtracted and spatially detrended Processed Signal intensities. To reduce the effect of noise in the miRNA microarrays data, we aggregated probes signals to have a more robust measure of expression, by computing the median of the intensity values (gProcessedSignal) of the set of probes corresponding to each miRNA. The samples were then normalized by the quantile method.
Tobacco-induced microRNAs and mRNAs profile alterations in human spermatozoa: a preliminary study for further knowledge of toxical spermatogenesis impairment
